Note: Descriptions are shown in the official language in which they were submitted.
P-7~1
SELECTIVE AGENT FOR GROUP A STREPTOCOCCI
The present invention relates to rapid detection
and presumptive identification of group ~ strepto-
cocci (Streptococcus p~ ~nes) by means of a selec-
tive blood agar medium.
BACKGROUND OF THE INVENTION
__ _
Streptococci, a genus of the family Lactobacil-
laceae are well distributed in nature. Some strains
of streptococci are pathogenic to man and/or animals,
whereas others may exist as generally harmless
parasites. Streptococci are classified (Lancefield
classification system) according to the presence
of a type of carbohydrate known as the C substance,
and at least 13 groups, designated A through O with
two alphabetic omissions are known. Of these groups,
group A ( _ pyogenes) is almost always responsible
for human infections, although other groups, in
certain circumstances, might also be pathogenic.
Group A streptococci (GAS) are responsible for
such diseases as scarlet fever, rheumatic fever,
glomerulonephritis, pharyngitis and puerperal spesis;
hence, identification of the presence of GAS can be
very significant in diagnosing a disease and se-
lecting an appropriate course of treatment.
According to present methods of detection of
GAS, a specimen is obtained by means of a cotton or
polyester swab applied to the infected area, such as
the pharynx. The swab is then used to inoculate a
suitable medium containing mammalian blood in order
to detect the characteristic complete or "beta"
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P-741
hemolysis which appears around colonies of GAS.
However~ a throat culture usually contains other
microorganisms which may be present in a much greater
number so that their growth obscures the hemolysis
produced by GAS~ The presence of bacteria resistant
to bacitracin also prevents the determination of
bacitracin susceptibility on the plate inocluated
with the specimen. Therefore, prior art methods
require isolation of the beta-hemolytic organisms in
lQ pure culture to determine bacitracin susceptibility
as an aid to presumptive identifica~ionO Thus, two
or more days may be required before information is
available to initiate appropriate therapy.
Although a number of ~anti-bacterial agents are
known to which GAS are resistant, there is no single
anti-bacterial agent or known combination of anti-
bacterial agents which are fully selective for GAS,
i~e., which will inhibit the growth of most other
microorganisms. Non-group A streptococci and other
microorganisms may, therefore, grow sufficiently on
existing selective media so as to make it difficult
to detect the characteristic beta hemolysis produced
by GAS. Furthermore, if other beta-hemolytic bac-
terial species are present, false positive results
may be obtainedO
Current methods for the detection of GAS are
relatively reliable, often achieving upwards of 90
per cent accuracy but require isolation in pure
culture of the beta hemolytic streptococci in order
to determine bacitracin susceptibility for presump-
tive identification. This takes two to-three days
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longer than the method of the present inventionO
Because of the poor selectivity of existing media,
greater skill is required to prevent error in diagno-
sis. A more selective medium would reduce interpre-
tive errors and would result in earlier .identifica-
tion and hence earlier institution of appropriate
therapy to prevent the serious sequelae of infection
by GAS.
SUMMARY OF THE INVENTION
An agent is provided for selectively culturing
GAS. The agent is a mixture of 1) a polymyxin or an
aminoglycoside, 2) a sulfonamid~, such as sulfa-
methoxazole~ 3) trimethoprim, and 4) crystal violet.
When the agent is incorporated in a mammalian blood-
containing culture m~di.um, bacteria other than GAS
are substantially eliminated, thus, permitting an
unobscured observation of the beta hemolysis sur-
rounding colonies of GAS. As a result of the sub-
stantial improvement in selectivity afforded by the
selection agent of the present invention, presumptive
identification of GAS based on susceptibility to
bacitracin (0.04 unit disc) and beta hemolytic
zones surrounding colonies of these organisms is
obtained within 18 to 24 hours after inoculation with
the specimen~
DETAILED DESCRIPTION OF THF PREFERRED EMBODIMENTS
_._ __ _
In accordance with the present invention,
~r
120S~2~ P- 741
--4--
improved detection of ~AS is achieved with a selec-
tion agent that permits selective growth of GAS, (S.
~yogenes), substantially inhibiting the growth of
other microorganisms which are likely to be present
in a biological specimen, e.g., a specimen taXen from
a human. This improved selectivity allows the deter-
mination of bacitracin (0.04 units) susceptibility or
serological testing o~ the primary isolation plate.
All of these components are individually known for
their anti-bacterial properties; however, this
cornbination of anti bacterial components has not been
previously described, and it is found that the
selection agent of the invention gives very signifi-
cantly improved selectivity for GAS. In particular,
when the selection agent is incorporated into a
blood-contaning culture plate on which a human
biological specimen containing GAS is inoculated,
very clear beta hemolysis patterns are observed in
the region of GAS growth, and vexy little extraneous
growth from other microorganisms is present on the
plate. This also permits, in most casesr the pre-
sumptive identification based on bacitracin suscep-
tibility and confirmatory identlfication by serologi-
cal grouping tests within 24 hours a~ter inocluation
with the specimen.
Polymyxins are complexes produced by Bacillus
polymyxa, and these complexes and their sulfates are
known to have antibacterial properties. Polymyxin
takes several forms including A, B, C, D, E, K, M and
P. Preferably, polymyxin E sulfate (colistin sul-
fate) is one of the components of the selection
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agent. As a substitute for polymyxin sulfate, an
aminoglycoside, such as gentamicin, amikacin, neo-
mycin or tobramycin can be used. These components
are known to be active against coliform bacilli and
other Gram-negative bacteria.
Another component of the selection agen~ is a
sulfonamide, such as sulfamethoxazole, sulfisoxazole,
sulfadiazine, or any other which demonstrates syner-
gistic activity in combination with trimethroprim
against alpha Streptococcus and is sufficiently
soluble to be used in an aqueous-culture medium. The
preferred sulfonamide is sulfamethoxazole. Sulfona-
mides are known to inhibit both Gram-positive and
Gram-negative bacteria, but streptococci are mostly
resistant.
Another component of the selection agent is
trimethoprim or trimethoprim lactate. Trimethoprim
is effective in inhibiting both Gram-positive and
Gram-negative bacteria, but in combination with a
sulfonamide, it exhibits a synergistic effect, so
that many organisms that are not inhibited by
either alone are inhibited when both are present.
The combination of sulfamethoxazole and trimethoprim
has been used previously in culture media for selec-
tive growth of GAS, but is not effective in in-
hibiting staphylococci.
The final component i5 crystal violet also known
as gentian violetD which is a mixture of penta~ and
hexamethyl p-rosanaline chloridesO This component is
known to inhibit neomycin resistant staphylococci.
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The selection agent according to the invention
typi~ally comprises between about 30.0 and abou~ 50~0
mg per liter of a culture medium. The sulfonamide is
generally the antibacterial component of greatest
amount by weight, typically being used in amounts of
between about 15 and about 30 mg per liter of the
culture medium. Polymyxin sulfate is used in amounts
of between about 10.0 and 20 0, preferably 15.0 mg
per liter of culture medium. If an aminoglycoside is
substituted for the polymyxin, it is present at
between about 5.0 and 30.0 mg per liter, depending on
which aminoglycoside is used. Trimethoprim is used
at about 5 per cent of the weight of the sulfonarnide,
i.e., pr~ferably between abou~ 0.74 and about
1.5 mg per liter of culture medium. Crystal violet
is used at between about 0.1 and about 0.3 mg per
liter of culture medium.
The culture medium to which the se~ection agent
is added includes an organic nitrogen source, such as
peptones, mammalian blood, salt and a gelling agent,
preferably agar. Peptones are present in amounts of
about 15 to about 30 gm per liter. The preferred
peptones are pancreatic digest of casein and a papaic
digest of soybean meal J with the casein digest
being present in amounts of from about 10 to about
20, preferably 1~, gm per liter and soybean meal
digest being present in amounts of from about 3 to
ab~ut 8, preferably 5, gm per liter. Agar in amounts
of from about 13 to about 20, preferably 15, gm per
liter provides a gel of appropriate firmness.
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Sodium chloride is provided in amounts of from
about 2 to about 8 gm per liter and preferably about
5 gm per liter. The salt concentration is important
for proper osmolality of the gel, preventing prema-
ture hemolysis of the blood~
Mammalian blood, which is present in amounts of
from about 4.0 to abo~t 10.0 volume per cent, is
derived from a variety of mammalian sources, in-
cluding sheep and rabbit. The blood is defibrinated
for use in a culture medium to prevent clotting~ The
blood, as stated above, is an indicator for hemolytic
organisms, such as GAS, producing charac-teristic
hemolysis in the present of such org~nisms. The
occurrence of bet~a hemolysis around colonies growing
on a culture plate having the selection agent that
substantially inhibits the growth of other hemolytic
microorg2nisms is a strong indication of the presence
of GAS.
Additional confirmation of the presence of GAS
on a blood culture plate containing the selection
agent is the ab.sence of beta hemolytic colonies in a
region containing bacitracin, an antibiokic produced
by a member of the Bacillus subtilis group. Bacitra-
cin~impregnated discs (0.04 units~ are commercially
available for placement on culture plates to apply
the antibiotic in limited regions of the plates.
Furthermore, definitive serological identification
can be obtained on a culture plate containing the
selection agents by means oE commercially available
~its.
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Culture media containing the selection agent
provide exceptional inhibition of normal microorga
nismst especially the viridans streptococci, which
are the predominant organisms present in most throat
culturesO While inhibition of viridans streptococci
is accomplished with a mixture of sulfamethoxazole
and trimethoprim alone, the selection agent of the
invention also provides improved inhibition of
staphylococci, other streptococci and Gram-negative
bacteria, including neisseria species and Pseudomonas
aeruginosa. In addition, the highly selective nature
of the selection agent of the invention permits the
presumptive identification of GAS (through the
appearance of hemolytic colonies on the plate
and the absence of hemolytic colonies in the region
of a bacitracin disc~ and definitive serological
grouping substantially earlier than is possible with
prior art formulations, e.y~, 18 to 24 hours as
compared to 42 to 72 hours.
The invention will now be described in greater
detail by means of specific examples.
Example 1
A group A streptococcus selective medium is
prepared having the following ingredients:
Per Liter of
Ingredient Purified Water
~ . _
Pancreatic Digest oE Casein 15.0 g
Papaic Digest o~ Soybean Meal 500 g
Sodium Chloride 5~0 g
Agar15.0 g
Colistin Sulfate 15.0 mg
Sulfamethoxazole 23.75 mg
Trimethoprim 1.25 mg
Crystal Violet 0.2 mg
Sheep Bl03d~ Defibrinated50.0 mL
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The ingredients, excluding the sheep blood, may
be conveniently mixed together to produce a homo-
genous powder~ The powdered medium is hydrated with
one liter of distilled or deionized water and boiled
until all ingredients are dissolved and the medium is
clear. The medium is then cooled to 45 to 50~C in a
water bath, and ~he defibrirlated sheep blood is
added. The medium is mixed by agitation and 18 to
20 rnl is dispensed into 100 mm Petri dishes according
to standard bacteriologic practice~ After this
medium is gelled, the dishes are placed in a suitable
container, such as a sleeve of plastic film (cello-
phane, polyethylene, NylonR, etc.), and are stored
at 2 to 8C until ready for use. The plates may be
stored in this manner for up to 12 weeks.
A throat swab specimen from a patient who was
subsequently shown to contain GAS is inoculated onto
the above described plated medium and a plate of
non-selective blood agar, according to routine
microbiological procedure. The inoculum is then
spread out with a sterile (flamed) bacteriological
inoculating loop so as to obtain isolated colonies.
In the area of heaviest inoculation, several cuts are
made into the agar with the loop and a 0~04 unit
2S bacitracin disc is placed a few cen~imeters away Erom
the cut~ but still in the heaviest inoculated area.
The plate is then incubated at 35 ~ 2C in an atmo
sphere of 3 to 8% carbon dioxide for 18 to 24
hours.
Upon examination, the beta hernolytic GAS colo-
nies on the non-selective blood agar plate are
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difficult to distinguish from the heavy growth of
normal throat flora also present on the medium~
Sensitivity to the bacitracin disc is also difficult
to determine. The plated medium containing the
selective agent, however, provides excellent suppres-
sion of the normal throat flora a~d permits unob-
scured observation o the beta hemolysis of GAS~ A
zone of inhibition surrounding the bacitracin disc is
clearly evident, thus resulting in the presumptive
identification of the beta hemolytic growth as GAS
within only 18 to 24 hours after inoculation with the
specimen.
Exam~le 2
The medium (A) prepared in Example 1 above is
compared with media (B-E) in an identical manner
except with or without certain selective components
as indicated by the~ pluses or minuses in the table
below. The media B-E are commercially available.
A B~l) C(2) D(3) E(4)
Colistin Sulfate ~ - - +
Sulfamethoxazole ~ +
Trimethoprim
Crystal Violet
Neomycin Sulfate - ~ +
Nalidixic Acid - - + ~ _
~1) Neomycin Blood agar
(2) Neomycin/Nalidixic Blood Agar
(3) CNA Agar
(4) SXT Blood Agar
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The concentrations of each cornponent where
present are equal to the amounts as specified in
Example 1 above~ The concentration of neomycin
sulfate in media B and C is 30 mg per literO The
concentration of nalidixic acid is 15 mg per liter in
medium C an~ 10 mg per liter in medium D.
The media are evalua~ed with specific cultures
to determine relative performance with respect to the
recovery of GAS and the inhibition of non-GAS present
in normal throat specimens~ Results are indicated in
Table 1.
TABLE 1
A B C D E
Recovery of GAS XXX XXXX XXXX XXXX XXX
Beta-hemolysis of GAS XXX XXXX XXXX XXXX XXX
Inhibition of group B
streptococci XXX - X X XXX
Inhibition of groups C
and G streptococciXXXX - X X XXXX
Inhibition of viridàns
streptococci XXXX - - - XXXX
Inhibition of other
beta~ or non hemolytic
streptococci XXX - X X XXX
Inhibitio~ of Neisseria XXXX XXXX XXXX XXXX XX
Inhibition of Pseudomonas XXXX - XX XXXX XX
.
Inhlbition of other
gram negative species XXXX XXXX XXXX XXXX XXXX
Inhibition of 5. aureus XXXX XXXX XXXX X XX
XXXX EXCELLENT
XXX GOOD
XX FAIR
X TRACE
- NONE
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The results, as set forth in the above table,
illustrate the surprising improvement of the selec-
tion agent of ~he present invention over selection
agents known in the prior art.
EXAMPLE 3
In a study involving human throat specimens, the
selective medium of the invention (medium A in
Example 1~ and SXT Sheep Blood Agar (medium E in
Example 2) are compared with non-selective sheep
blood agar (control). Throat swab specimens are
inoculated onto each of the three media in accordance
wi th the procedure described in Example 1. Inocu
lated media are then incuba~ed for 18 ~o 24 hours at
35 -~ 2~C in an atmosphere of 3 to 8% carbon dioxide.
Of 460 random throat cultures, 117 are positive for
G~S with the media containing the selective agent of
the invention (,A), 130 with SXT Blood Agar (E) and
only 84 with the non-selective control as illustrated
in the table below~
A E Control
Cultures positive for GAS at 24 hours 103 80 32
Cultures positive for GAS at > 48 hours 14 20 52
TOTAL 117 100 84
Furthermore, of the 117 cultures positive for
GAS on the selective medium of the invention (A), 103
are presumptively identified as GAS by bacitracin
susceptibility within 24 hours. This represents a
significant improvement as compared to 80 of 100 on
SXT Blood Agar (E) and only 32 of 84 on the non-se~
lective control
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~13-
Although the invention has been described in
terms of certain preferred e~bodiments, modiications
obvious to one with ordinary skill in the art may be
made without departin~ from the scope of the inven-
tion. For example, while the invention sets forth
four groups of components to be included in a medium,
it is contemplated that additional components might
be added, further enhancing medium selectivity.
Various features of the :invention are set forth
in the following claims.
